1 research outputs found
Detection and Identification of the Vibrational Markers for the Quantification of Methionine Oxidation in Therapeutic Proteins
Methionine
oxidation is a major degradation pathway in therapeutic
proteins which can impact the structure and function of proteins as
well as risk to drug product quality. Detecting Met oxidation in proteins
by peptide mapping followed by liquid chromatography with mass spectrometry
(LC–MS) is the industry standard but is also labor intensive
and susceptible to artifacts. In this work, vibrational difference
spectroscopy in combination with <sup>18</sup>O isotopic shift enabled
us to demonstrate the application of Raman and FTIR techniques for
the detection and quantification of Met oxidation in various therapeutic
proteins, including mAbs, fusion proteins, and antibody drug conjugate.
Vibrational markers of Met oxidation products, such as sulfoxide and
sulfone, corresponding to SO and C–SO stretching
frequencies were unequivocally identified based <sup>18</sup>O isotoptic
shifts. The intensity of the isolated νC–S Raman band
at 702 cm<sup>–1</sup> was successfully applied to quantify
the average Met oxidation level in multiple proteins. These results
are further corroborated by oxidation levels measured by tryptic peptide
mapping, and thus the confirmed Met oxidation levels derived from
Raman and mass spectrometry are indeed consistent with each other.
Thus, we demonstrate the broader application of vibrational spectroscopy
to detect the subtle spectral changes associated with various chemical
or physical degradation of proteins, including Met oxidation as well
as higher order structural changes